Blood-feeding mosquitoes transmit a diversity of pathogenic organisms, devastating human health and the global economy. The prevention of mosquito-borne diseases with insecticides has been very successful in the past, but it was also recognized as damaging to the environment. Furthermore, it became increasingly expensive and ineffective with the spread of insecticide-resistant mosquitoes. New opportunities for disease control arose with post-genomic progress in vector biology. For example, genome wide screening has revealed that mosquito metabolism is deficient in the synthesis of half of the proteinogenic amino acids, which therefore must be acquired from larval habitats or adult blood meals and properly distributed in organism to maintain mosquito life cycle. During the previous funding period we discovered fundamental differences in the molecular basis of essential amino acid absorption and redistribution in mosquitoes vs. other organisms. Specifically, we have cloned and characterized a representative set of mosquito-specific Nutrient Amino acid Transporters (NATs) from the Neurotransmitter Sodium Symporter (NSS or SLC6) family. We determined that NATs comprise the major mechanism for the active absorption and redistribution of all essential amino acids in mosquitoes. Hence, genetic or pharmacological suppression of NAT function is expected to be critical for mosquito development and oogenesis, which demand massive accumulation and intensive systemic traffic of essential amino acids. Next, we propose to explore the integrative, regulatory, and structural aspects of the unique NAT-SLC6 population and analyze the impacts of pharmacogenetic NAT suppression in two model vector mosquitoes, Anopheles gambiae and Aedes aegypti. This project will be accomplished through the pursuit of four specific aims: (1st) To explore NAT regulation in mosquito nutrition, development, and neuronal functions;(2nd) To reveal effects of RNAi-induced NAT silencing on larval nutrition and development;(3rd) To develop a computational model of NAT interactions with substrates and inhibitors in silico;and (4th) To determine the impacts of pharmacological NAT inhibition in situ and in vivo, and combine these approaches as an innovative NAT-SLC6 drug discovery strategy. In pursuit of these aims we will: (i) Reconstruct spatial and developmental profiles of NAT expression in mosquitoes under different nutrient conditions via innovative parallel molecular labeling and quantification techniques;(ii) Accomplish 3D homology model-driven predictions of NAT substrates and inhibitors in silico;(iii) Complete in situ assays of selected NAT pharmacophores using heterologous expression systems;and (iv) Evaluate the molecular and physiological efficacies of NAT suppression by monitoring NAT expression in parallel with survival and development of mosquito larvae after injection-mediated RNAi silencing and feeding-mediated pharmacological inhibition. The anticipated results of this work will fill major gaps in our understanding of essential amino acid absorption in vector mosquitoes and develop the rationales for targeting NAT-SLC6s in vector control applications.